Studies of the interaction of Escherichia coli YjeQ with the ribosome in vitro
Studies of the interaction of Escherichia coli YjeQ with the ribosome in vitro
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DOI：
10.1128/jb.186.5.1381-1387.2004
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发表时间：
2004-03-01
影响因子：
3.2
通讯作者：
Brown, ED
中科院分区：
文献类型：
--
作者：
Daigle, DM;Brown, ED
大肠杆菌YjeQ代表了一组保守的细菌特异性核苷酸结合蛋白，其生理功能未知，已被证明是大肠杆菌生长所必需的。coli和Bacillus subtilis。该蛋白先前已被表征为具有缓慢稳态GTP水解活性（8 h（-1））（D. M.戴格尔湖罗西A. M.贝尔吉斯湖Aravind，E. V. Koonin和E. D. Brown，Biochemistry 41：11109-11117，2002）。本文报道了E.发现大肠杆菌与来自细胞提取物的核糖体共纯化。然而，每个细胞的蛋白质拷贝数相对于核糖体的数量是低的（YjeQ拷贝与核糖体的比率，1：200）。在体外，重组YjeQ蛋白与30 S核糖体亚基强烈相互作用，并且在存在不可水解的GTP类似物5 '-鸟苷酰亚氨基二磷酸（GMP-PNP）的情况下，用盐洗涤揭示的该相互作用的严格性最高。同样，与30 S亚基的关联导致YjeQ GTdR活性的160倍刺激，其在化学计量量的核糖体下达到最大值。YjeQ的N-末端截短变体揭示了预测的OB-折叠区域对于核糖体结合和GT3刺激是必需的，并且它们表明N-末端肽（YjeQ中的氨基酸1至20）对于YjeQ与30 S亚基的GMP-PNP依赖性相互作用是必需的。两者合计，这些数据表明，YjeQ蛋白参与了鸟嘌呤核苷酸依赖的相互作用与核糖体和牵连这种保守的，必要的GTdR作为一种新的因素在核糖体功能。
Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E. coli and Bacillus subtilis. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h(-1)) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry 41:11109-11117, 2002). In the work reported here, YjeQ from E. coli was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.